Steroid synthesis in the adrenal gland of the sea snake Hydrophis cyanocinctus: The metabolism of exogenous precursors

Sea snake (Hydrophis cyanocinctus) adrenal glands (whole homogenates, preincubated minces, or mitochondrial preparations) were incubated in vitro with exogenous radioactive precursors. Hydrophis adrenal tissue was capable of synthesizing 17-deoxycorticosteroids from exogenous cholesterol, pregnenolone, progesterone, and DOC, but not from sodium [¹⁴C]-acetate. Products identified after incubation were pregnenolone, progesterone, 11β-hydroxyprogesterone, DOC, corticosterone, 18-hydroxycorticosterone, and aldosterone. The major product was corticosterone with lesser quantities also of 18-hydroxycorticosterone and aldosterone. In the case of the mitochondrial preparation 11β-hydroxyprogesterone predominated. No evidence for the biosynthesis of cortisol from cholesterol was found. Two types of kinetic incubation were employed: One sampled the incubation medium alone, while the other sampled both medium plus tissue. It was concluded that sampling the medium only did not allow the identification of the biosynthetic pathways operating in vitro. However, from sampling both the medium and the tissue it was concluded that both the C21-C11 and C11-C21 sequences of hydroxylation operated in the conversion of progesterone to corticosterone. The data contrast with those obtained from previous studies on cobra adrenal tissue, particularly with regard to the ability of sea snake adrenals in vitro to 18-oxygenate exogenous precursors.     

The pituitary--adrenal axis in the sea snake, Hydrophis cyanocinctus daudin.

The sea snake Hydrophis cyanocintus was subjected to conventional pharmacological tests of pituitary-adrenal function. Administration of synthetic ACTH resulted in adrenal hypertrophy, enhanced Δ⁵-3β-hydroxysteroid dehydrogenase (3β-OHSD) activity and raised peripheral titres of corticosterone and aldosterone to 300% those of controls. Metopirone treatment resulted in adrenal hyperplasia and enhanced 3β-OHSD activity, but aldosterone titres were unchanged despite a 90% reduction in corticosterone titre. Dexamethasone administration was followed by adrenal atrophy and diminished 3β-OHSD activity. Titres of both corticosteroids were much lowered. Despite such marked variations in adrenal function, no alteration in plasma ion concentrations or total osmolarity could be detected. These and earlier data suggest that separate regulatory mechanisms for corticosterone and aldosterone may exist in the sea snake, and such a differential regulation may be of physiological importance.     

Clearance of corticosteroids in yearling coho salmon, Oncorhynchus kisutch, in fresh water and seawater and after stress

The metabolic clearance rate of corticosteroids determined after a single injection of [3H]cortisol was higher in fish acclimated to seawater (SW) than in those acclimated to fresh water (FW). Uptake and retention of corticosteroids in liver, gill filaments, and gall bladder bile were greater in SW than in FW fish. The stress of long-term (5 days), but not short-term (12 hr), continuous confinement apparently increased the clearance rate of corticosteroids in both FW and SW fish. Chronic, but not acute, administration of exogenous cortisol, yielding cortisol titers close to the physiological range, appeared to increase the clearance rate of corticosteroids in FW fish.     

Changes in plasma corticosteroids during smoltification of coho salmon,Oncorhynchus kisutch

Plasma corticosteroid concentrations were determined by competitive protein binding assay in coho salmon, Oncorhynchus kisutch, during smoltification. Between January and April plasma corticosteroid levels decreased from 8.6 to 3.5 ng/ml, but increased to 24.0 ng/ml by the end of May in smolts retained in fresh water. This nearly eightfold increase in corticosteroids occurred as plasma thyroxin levels declined. Thus plasma corticosteroids appear to be increasing during the period when coho salmon normally migrate to sea.     

Bioassay for salmon prolactin using hypophysectomized Fundulus heteroclitus

A bioassay for salmon prolactin (PRL) is described. This assay which is based on the sodium-retaining action of PRL in the hypophysectomized killifish, Fundulus heteroclitus, has proved to be rapid, sensitive (250 pg PRL per gram of fish), and specific. The procedure has been used to characterize the biological activity of a highly purified PRL from the pituitaries of the chinook salmon, Oncorhynchus tschawytscha, and a similar PRL isolated (by acid buffer polyacrylamide disc gel electrophoresis) from pituitaries of coho salmon (O. kisutch) (MW ca. 22,000; isoelectric point >9).     

Fine structure of the corpuscles of stannius of the trout, Salmo gairdneri: Structural changes in response to increased environmental salinity and calcium ions

These studies confirm that there are two cell types, C₁ and C₂, in the corpuscles of Stannius (CS) of the trout, Salmo gairdneri, both of which have the appearance of protein-or peptide-secreting cells. C₁ cells have large secretory granules, up to 1.0 μm in diameter, while the C₂ cells have small granules up to 0.5 μm. In seawater (SW)-adapted trout the C₂ cell granules are significantly larger than those in freshwater (FW)-adapted trout. In fresh water the C₁ cells are relatively inactive and the C₂ cells are mainly sparsely granulated and actively secreting. The converse situation is found in seawater-adapted trout in which the C₁ cells are very active and the C₂ cells are relatively inactive and contain stored granules. During adaptation to SW from FW some C₁ cells develop extensive rough endoplasmic reticulum and Golgi but other C₁ cells degenerate and disintegrate apparently releasing the entire cell contents, including granules, into the capillary lumena. Protein may be released from these granules in capillaries by digestive enzymes from lysosome-like vesicles. In both adapted FW and SW corpuscles the active cells appear to release their granules by exocytosis. The appearance of C₁ cells of fish transferred from FW to calcium-enriched fresh water is similar to that of fish transferred to dilute SW, suggesting that these cells respond to high levels of external calcium, while the C₂ cells are active in media of low ionic and osmotic strength.     

Pituitary responses to calcium deprivation in a euryhaline teleost, Fundulus heteroclitus, adapted to artificial sea water

Male killifish adapted to calcium-free sea water developed hypocalcemia. In the pituitary the PAS-positive cells of the pars intermedia (PIPAS cells) displayed a strong reaction, increasing in numbers and becoming enlarged and more active. A very minor response was shown by a small minority of the prolactin cells which became marginally activated. No other adenohypophysial cell type responded to calcium deprivation, and no response was detected in the neurohypophysis. It is suggested that the PIPAS cell may secrete a hypercalcemic hormone which plays a major role in calcium regulation in sea water.     

Calcitonin content in the ultimobranchial gland of the snake,Elaphe climacophora: Comparison of adults,young, and hatchlings

Hypocalcemic activity of the ultimobranchial gland of Elaphe climacophora was determined using the rat bioassay. The activity of one ultimobranchial gland of the adult females corresponds to that of 55 mU (MRC) of salmon calcitonin. The activities of one gland of the young and the hatchling are comparable to those of 17.5 and 18.0 mU of salmon calcitonin, respectively. The activity per unit body weight is extremely high (2,700 mU/kg body wt.) in the young and the hatchlings in comparison with that in the adults (460 mU/kg body wt.).     

Correlations between integument structure and ecdysteroid titers in fifth-stage nymphs of the tick, Ornithodoros moubata (Murray, 1877; sensu Walton, 1962)

The integument structure of fifth-stage nymphs of the tick Ornithodoros moubata was studied during the molting processes which are induced by the blood meal. In parallel, the ecdysteroid titers in hemolymph and in extracts from whole animals were determined by a radioimmunoassay (RIA). Between Days 2 and 3 after the meal (a.m.) a few procuticle lamellae were deposited and the mitotic period initiated. Ecdysteroid titers were low during the first 3 days a.m. They began to increase between Days 3 and 4 a.m. with the mitotic period ending. The titers rose sharply when apolysis and formation of the exuvial space took place (Day 4–5 a.m.). Highest ecdysteroid concentrations were measured during the deposition of the new epicuticle (Day 5–6 a.m.). Concomitant with the beginning of procuticle deposition and digestion of the old cuticle (Day 6 a.m.) the titers began to drop and reached low values again shortly before ecdysis (Day 9–10 a.m.). A preliminary chemical identification by thin-layer and high-performance liquid chromatography revealed that the majority of the RIA-positive ecdysteroids were composed of ecdysone and mainly ecdysterone. This was confirmed by gas chromatography of the silylated ecdysteroids. The results demonstrate that the titer changes in relation to the molting events in both ticks and insects appear to be very similar. It is thus likely that ecdysteroids are important in the control of tick molting.     

The effects of prolactin and TSH on thyroid function in Fundulus heteroclitus

Treatment with ovine prolactin significantly reduces serum T₄ in Fundulus heteroclitus, both in control (P < 0.05) and TSH-stimulated fish (P < 0.01), without affecting thyroidal radioiodine uptake. Prolactin also reduces the loss of radioiodine from F. heteroclitus (P < 0.001). This loss is markedly increased by ovine TSH (P < 0.001), but only in the absence of prolactin. Although other explanations are possible, prolactin is probably goiterogenic in F. heteroclitus, lowering thyroidal secretion of T₄ as well as inhibiting the peripheral effects of this hormone.     

The effects of hypophysectomy and replacement therapy with prolactin,cortisone, or their combination on the blood of the black bullhead Ictalurus melas

Experiments are done on intact and hypophysectimized Ictalurus melas maintained in approximately isotonic saline. Serum Na⁺ decreases after hypophysectomy but serum Cl^? is not affected. Prolactin increases both serum Na⁺ and Cl^? in intact fish, but only Na⁺ increases in hypophysectomized recipients. Cortisone decreases serum Na⁺ in hypophysectomized fish but increases serum Cl^?. When prolactin and cortisone are administered together, the Na⁺-increasing effect of prolactin is dominant, while the Cl^?-increasing effect of cortisone is greatly enhanced. It is concluded that the two ions are regulated separately and that both hormones are necessary to maintain normal levels in hypophysectomized recipients. Serum Ca²⁺ is higher in adult females than in males and low in juveniles, irrespective of treatment. Serum inorganic P is lower in juveniles than in adults and is unaffected by hypophysectomy. Serum organic P increases in hypophysectomized fish receiving cortisone, perhaps in correlation with the increase in serum proteins. Saline injections are hyperglycemic in intact fish but prolactin injections are hypoglycemic, as is hypophysectomy. Serum cholesterol tends to increase after hypophysectomy and injections of prolactin and/or cortisone result in a significant decrease. No sexual differences in serum proteins are detected in adult, postspawning fish and hypophysectomy has no effect in September, but low levels characterize winter fish at low temperature. Cortisone increases serum proteins in hypophysectomized fish in winter, but otherwise no hormonal effects are observed. Prolactin significantly lowers the pH of the blood, alone or in combination with cortisone.     

Immunocytochemical localization of thyroglobulin in the transforming endostyle of anadromous sea lampreys, Petromyzon marinus L., during metamorphosis

Light and electron microscopic immunocytochemistry was used to detect the localization of thyroglobulin (TG) in the transforming endostyle of the anadromous sea lampreys, Petromyzon marinus L., during metamorphosis. TG is found within the transforming endostyle throughout metamorphosis (stages 1 to 7) and in early stages (1–2) staining for TG is similar to that observed in the endostyle of the ammocoete. By stage 3, most of the TG is found in large bodies that accumulate within type 3 cells. At stage 4 immunostaining for TG is found within the epithelial cells of the transforming endostyle and in the lumina of some of the first formed follicles. Most of the follicular lumina contain TG by the end of metamorphosis (stage 7). TG in the young parasitic adult lamprey is localized within the follicular lumina, colloid droplets, and occasionally in large bodies found in some of the follicular epithelial cells.     

Serum gonadotropin and steroids associated with breeding activities in the green sea turtle,Chelonia mydas: II. Mating and nesting in natural populations

Circulating levels of gonadotropins (FSH and LH) and sex steroids (estrogen, testosterone, and progesterone) were examined in mating and nesting animals from three breeding aggregations of green sea turtles (Chelonia mydas). Copulating males were characterized by relatively low levels of both gonadotropins and high androgen levels, but the latter did not differ from values previously observed in prebreeding males. Females showed significant changes in all hormones studied, except estradiol. The patterns of changes in gonadotropins and steroids as well as their absolute values were similar among the three natural breeding aggregations and captive turtles studied previously. Serum or plasma LH showed slight but variable increases between mating and nesting animals, whereas FSH was consistently elevated at the time of nesting. Field studies demonstrate that this rise in FSH is a transient surge of no more than a few hours' duration: FSH does not rise until the middle of the nesting process, when oviposition begins, and FSH declines before or immediately after oviposition is completed. Thus, this peak in FSH apparently does not initiate nesting behavior. Testosterone did not differ between mating and nesting in females, but it dropped significantly in the first days following nesting (i.e., in the internesting interval when ovulation occurs). Progesterone values were also similar between mating and nesting samples, but this steroid along with LH frequently showed a pronounced elevation in the 1–2 days postnesting; their elevation was considered to represent the “ovulatory surge”. Since this progesterone and LH surge only occurs if a nesting turtle is allowed to oviposit, the presence of shelled oviducal eggs probably suppresses ovulation.     

Synchronous maturation of the female reproductive system and of the ovulation hormone-producing system in Lymnaea stagnalis

The intercerebral commissures (COMs) of young snails, which are in the male stage (10–15 mm shell height), contain only small quantities of ovulation hormone (CDCH): 0.6 OIU · COM^?1 · g^?1 snail (1 ovulation inducing unit = threshold dose for ovulation in adult recipients). During the subsequent period of female maturation the quantities of CDCH increase considerably up to a maximum of 47 OIU · COM^?1 · g^?1 snail at the onset of oviposition. In snails smaller than ca. 21 mm shell height oviposition cannot be induced by COM extract injections. It is concluded that maturation of the female reproductive system and of the ovulation hormone system occur simultaneously, and that both processes are stimulated by the hormone of the dorsal bodies (endocrine organs located upon the cerebral ganglia).     

Erythropoietin,testosterone, and thyroxine in the erythropoietic response of the snake, Xenochrophis piscator

The erythropoietic response of snakes was examined after injecting human urinary erythropoietin (Ep), testosterone propionate (TP), and l-thyroxine (T₄), separately and in combinations, into starved ophids. The effect of starvation was reflected by a decrease in the number of erythrocytes, a fall in hemoglobin concentration, and a decline in hematocrit. Statistically significant elevation of erythrocyte number, hemoglobin concentration, and hematocrit was observed at 24 hr following the administration of Ep + T₄, and Ep + TP + T₄ into starved ophids. The erythrocyte number was also increased by T₄ treatment at 24 hr. Furthermore, while T₄ and Ep individually increased the red blood cell number at 168 hr, T₄, TP + T₄, and Ep + TP + T₄ elevated the hemoglobin concentration and Ep + T₄ and Ep + TP + T₄ increased the hematocrit value. It is suggested that the influence of any one of the hormones utilized in the present study on blood morphology of fasted snakes depends to a greater extent on the presence or absence of the other hormone(s).     

An inexpensive servo-control system for regulating microvascular perfusion pressures in small animals

A simple and inexpensive system for controlling the pressure perfusing the rat cremaster muscle is described. The system utilizes an inflatable cuff-type vascular occluder that is placed around the sacral aorta and is inflated using a syringe pump. The syringe pump is turned on and off by a feedback comparator which senses the pressure downstream of the occluder, and varies the degree of occlusion until the downstream pressure equals a desired setpoint value. Although very simple, this system provides a useful and effective means of altering pressure within the cremaster muscle, and could easily be adapted to other microvascular preparations.     

Effects of eyestalk removal on cholesterol uptake and ecdysone secretion by crab (Cancer antennarius) Y-organs in vitro

Y-Organs and control tissues from intact (intermolt) and 48-hr de-eyestalked Cancer antennarius donors were cultured for 12 and 24 hr in crustacean saline supplemented 10% with crab serum and containing [14C]cholesterol. Under these conditions, Y-organs took up significantly more [14C]cholesterol than ovary or muscle, and Y-organs from 48-hr de-eyestalked crabs took up threefold more than Y-organs from intact crabs. The labeled cholesterol of the culture medium was observed to bind rapidly to the lipoproteins of the serum supplement; subcellular fractionation of the activated Y-organs after incubation showed 59% of the label localized in the cytosolic fraction. The increase in cholesterol uptake did not result from a change in extracellular volume, and was not accompanied by a change in Y-organ total cholesterol. It was, however, accompanied by a greater than threefold increase in ecdysone secretion.     

Thyroidal influence on the lactate dehydrogenase isozyme pattern in pectoralis muscle of chick embryos

The effect of methimazole (MMI) treatment on the lactate dehydrogenase (LDH) isozyme pattern in pectoralis muscle of chick embryos was ascertained using polyacrylamide gel electrophoresis. Muscle extracts from 20-day embryos contained the five isozymes typical of most vertebrates, but 9 out of 10 extracts from 17-day embryos did not contain detectable levels of LDH 5. Muscle extracts from 20-day embryos treated with MMI for 6 to 10 days prior to death had less LDH 3, 4, and 5, compared to 20-day controls. Except for faint staining in the region of LDH 3, LDH 1 and 2 were the only isozymes detected in the extracts from 16 out of 21 MMI-treated embryos. The administration of single, low doses of thyroxine at 17 or 18 days of incubation resulted in detectable amounts of LDH 4 at 20 days in muscle from MMI-treated embryos; 7 out of the 25 muscles analyzed also had detectable amounts of LDH 5. The present report of thyroidal influence on muscle LDH isozyme pattern supports earlier observations which indicated that thyroid function is important to muscle growth and development during late embryogenesis in the chick.     

Aromatase activity in marsupial brain,ovaries, and adrenals

In contrast to numerous other vertebrate groups in which there is biochemical evidence for aromatase activity in brain tissues, estrogen synthesis was not detectable in the CNS of a North American marsupial, the Virginia opossum. In the present study, the occurrence of aromatase activity in marsupial brain and other tissues was reinvestigated using the Australian species, Macropus eugenii and Trichosurus vulpecula. Following incubation of tissue homogenates with [7-³H]androstenedione and an NADH-NADPH generating system, estrogen products were isolated and finally identified by methylation and crystallization to constant specific activity. No estrogen was detectable in samples of adult brain; however, the brain of wallaby pouch young conformed to the general vertebrate pattern. Not only were small amounts of authentic estrone synthesized, but aromatization was greatest in limbic areas and activity per unit protein in all regions progressively declined with age. The ovaries of Macropus and Trichosurus as well as the adrenals of Macropus were also capable of converting labeled substrate to estrone or estradiol-17β. This study provides the first definitive evidence for synthesis of conventional eutherian estrogens in Metatheria.     

Ovarian cholesterol dynamics in teleost Channa punctatus (Bloch): Relationship with reproductive cycle and response to gonadotropins

Thin-layer chromatography was employed to separate free and esterified cholesterol from total ovarian cholesterol in the teleost Channa punctatus. Results clearly showed that free cholesterol was almost double the concentration (5.37 ± 0.32 nmol/mg) of esterified cholesterol (2.67 ± 0.26 nmol/mg). An annual profile of free and esterified cholesterol determined per ovary showed that both altered in different months of the year although fluctuation was more prominent with free cholesterol. In contrast, the study in terms of per milligramovarian tissue, showed fluctuation of free cholesterol only. Among the pituitaries collected in different reproductive stages, prespawning pituitary extract had maximum depletive effect on ovarian total and free cholesterol in comparison to others. Ovine LH (1 μg/100 g body wt) could effectively induce the depletion of ovarian free cholesterol; the most remark-able effect was obtained with a similar dose of SG-G100 but there was insignificant alteration of esterified cholesterol. In hypophysectomized fish, SG-G100 in increasing doses (1 to 10 μg/100 g body wt) evoked a linear response in regard to the ovarian free cholesterol depletion. These findings contrast strikingly with reports on the mammalian ovary.